Plasma microRNAs as potential biomarkers in early Alzheimer disease expression

The microRNAs (miRNAs) are potential biomarkers for complex pathologies due to their involvement in the regulation of several pathways. Alzheimer Disease (AD) requires new biomarkers in minimally invasive samples that allow an early diagnosis. The aim of this work is to study miRNAS as potential AD biomarkers and their role in the pathology development. In this study, participants (n = 46) were classified into mild cognitive impairment due to AD (MCI-AD, n = 19), preclinical AD (n = 8) and healthy elderly controls (n = 19), according to CSF biomarkers levels (amyloid β42, total tau, phosphorylated tau) and neuropsychological assessment. Then, plasma miRNAomic expression profiles were analysed by Next Generation Sequencing. Finally, the selected miRNAs were validated by quantitative PCR (q-PCR). A panel of 11 miRNAs was selected from omics expression analysis, and 8 of them were validated by q-PCR. Individually, they did not show statistically significant differences among participant groups. However, a multivariate model including these 8 miRNAs revealed a potential association with AD for three of them. Specifically, relatively lower expression levels of miR-92a-3p and miR-486-5p are observed in AD patients, and relatively higher levels of miR-29a-3p are observed in AD patients. These biomarkers could be involved in the regulation of pathways such as synaptic transmission, structural functions, cell signalling and metabolism or transcription regulation. Some plasma miRNAs (miRNA-92a-3p, miRNA-486-5p, miRNA-29a-3p) are slightly dysregulated in AD, being potential biomarkers of the pathology. However, more studies with a large sample size should be carried out to verify these results, as well as to further investigate the mechanisms of action of these miRNAs.

www.nature.com/scientificreports/ diagnosis of the pathology, and some miRNAs have been described as potential biomarkers for AD diagnosis and prognosis 15 . Some miRNAs showed good performance as biomarkers, mainly panels including different miRNAs might be dysregulated several years before the onset of disease symptoms 16 . Several panels were developed from plasma, serum or exosomes, showing their potential for a minimally invasive disease diagnosis [17][18][19] . In general, different results have been found in the literature, probably due to the lack of diagnostic biomarkers used in the classification of participants or due to the differential methodologies applied in sequencing or data processing. The aim of this work is to analyse the differential expression of a panel of miRNAs selected from sequencing analysis in plasma from AD and non-AD patients, evaluating their potential usefulness as biomarkers or in the development of therapeutic targets, as well as to study their potential implications in the course of the AD.

Material And Methods
Participants and samples collection. In this study, participants (n = 46) were aged between 50 and 80 years old and were classified into mild cognitive impairment due to AD (MCI-AD, n = 19), preclinical-AD (n = 8) and healthy controls (n = 19), according to the National Institute on Aging and Alzheimer's Association (NIA-AA) criteria. Briefly, the control group showed negative CSF biomarkers or amyloid PET, and normal neuropsychological assessment; the MCI-AD group showed positive CSF biomarkers or amyloid PET, and impaired neuropsychological assessment; the preclinical-AD group showed positive CSF biomarkers or amyloid PET, and normal neuropsychological assessment. Participants with other major disorders and those unable to complete the assessment were excluded. All participants were recruited at the Neurology Unit of the University and Polytechnic Hospital La Fe (Valencia, Spain) after signing informed consent, and plasma samples were obtained at the same time the lumbar puncture. The study protocol (2019/0105) was approved by the Ethics Committee (CEIC) of the Instituto de Investigación Sanitaria La Fe (Valencia, Spain). No randomisation was performed to allocate subjects in the study. No pre-registration was performed. The study is blinded as the classification of participants was performed by a person other than the experimenter. No sample calculation was performed. All methods were carried out in accordance with relevant guidelines and regulations. RNA extraction. RNA was isolated for RNA sequencing using the miRNeasy plasma kit (Qiagen, Germany) following the manufacturer's protocol. Briefly, 200 µL of plasma and 700 µL of QIAzol lysis reagent were incubated for 5 min at room temperature (RT). Then, 140 µL of chloroform were added and incubated at RT for 3 min and centrifuged at 1200 g (15 min, 4 °C). The aqueous phase was mixed in a new tube with 525 µL of ethanol and transferred to a RNeasy MinElute spin column followed by a centrifugation step at 10000 g (30 s, RT). The column was then washed with RWT buffer (700 µL) and RPE buffer (500 µL) and dried for 90 s at 10000 g. Finally, the elution step was performed with 15 µL of RNase-free water (13000 g, 1 min).
For PCR validation, RNA extraction was carried out in a similar way but including a previous step, which consisted on the addition of RNS spike-in before the protocol.

RNA sequencing method.
Construction of RNA libraries. The miRNA libraries were prepared from total RNA using the NEXTFLEX® Small RNA-Seq v3 Kit for Illumina Platforms (Bioo Scientific Corporation, Texas, USA). Briefly, the small RNA molecules were first ligated to the 3'-4 N adenylated adapters, taking advantage of the phosphate group at their terminal end, which allows the exclusive targeting of these molecules. Secondly, the 5'-4 N adapters were ligated. Later, reverse transcription of the molecule into cDNA was carried out. The generated cDNA fragments were then amplified and indexed by PCR using different barcode primers for each sample. Finally, a size-selective purification was carried out.
The quality control and concentration of the libraries were verified with the Agilent Technologies 2100 bioanalyser using highly sensitivity DNA chips (Central Unit for Research in Medicine (Universitat de València)). Subsequently, an equimolecular pool of each library was prepared for sequencing.
Sequencing on an Illumina equipment. Sequencing was carried out on the NGS NextSeq 550 platform (Illumina, San Diego, CA, USA) by single read sequencing of 50 cycles (1 × 50 bp).

Data analysis.
Pre-processing, quality control and normalization. NGS data (raw fastq files from sRNA sequencing) were processed following the standard protocol proposed by Cordero et al. 20 implemented in the function mirnaCounts from docker4seq package 21 with default parameters in R 22 . First, a sequence quality control check was generated using FastQC 23 and then cutadapt 24 program was used for the adapter trimming. Specifically, adapters and low-quality reads (Phred Score < 10) were trimmed and removed (44.014.980 reads). Once adapters were removed, sequence reads (219.207.246 good quality reads) were mapped against miRNA precursors from miRBase (v.21) 25 , using SHRIMP 26,27 , filtering out a total of 95,03% reads. Finally, miRNA quantification from the resulting 4,97% of mapped reads were generated using the function count Overleaps from GenomicRanges package 28 , resulting in a total of 9.799.858 miRNA counts in a total of 2.386 miRNAs. miRNAs selection. From the miRNAs identified in the pre-processing, quality control and normalization process, some of them were selected. Specifically, those miRNAs which showed a number of counts different from zero in at least 80% of the samples and that were corroborated in literature. Finally, the selected miRNAs were validated by means of qPCR in the same plasma samples. miRNAs validation by quantitative PCR. Quantitative PCR procedure. From the extracted RNA, retrotranscription and amplification steps were carried out following the manufacturer's recommendation www.nature.com/scientificreports/ (TaqMan Advanced miRNA Assays) [https:// tools. therm ofish er. com/ conte nt/ sfs/ manua ls/ 10002 7897_ TaqMa nAdv_ miRNA_ Assays_ UG. pdf]. Briefly, the protocol consisted of four steps. First, the addition of a polyA tail, after the adapter ligation, followed by the retrotranscription step, and then the specific miRNA amplification. Finally, samples were diluted, and real time PCR (RT-PCR) was carried out in duplicate using the thermocycler (ViiA7, Applied Biosystems, California, USA).
Statistical analysis. The number of counts obtained from RT-PCR were averaged for duplicates, discarding replicates with values within ± 2 counts from mean. Then, samples were normalized using the mean and standard deviation. The miRNAs detected in at least 80% of the samples and with a difference between replicates < 1 count were considered satisfactorily quantified. The effect of each biomarker on pathology was then analyzed by Bayesian models: the first model discriminates among control, MCI-AD and preclinical AD groups; and the second model discriminates between AD (preclinical AD, MCI-AD) and control groups. For these models, some parameters were calculated (estimate, which indicates the direction of the miRNAs levels; Odds Ratio; Percentage Inside Rope, which defines the percentage of the area that is within the region of practical equivalence (equivalent to null effect); probability of direction (PD), which indicates the probability that the effect has in a particular direction (indicated by the estimate). PD > 80% was considered significative).
Pathway analysis. The target genes of the differentially expressed miRNAs were studied using the miR data base (miRDB). The selected target genes were those with a target score ≥ 95. Then, the targets were classified according to cellular pathways and functions in order to analyze the implication in AD.

Ethics approval. The study protocol (2019/0105) was approved by the Ethics Committee (CEIC) from
Health Research Institute La Fe (Valencia, Spain).

Consent to participate.
Informed consent was obtained from all individual participants included in the study.

Research involving Human Participants and/or Animals. Yes, human participants.
Informed consent. All the participants were recruited in the Neurology Unit from University and Polytechnic Hospital La Fe (Valencia, Spain) after signing the informed consent.
These results are shown in Fig. 1, which depicts the PD and ROPE for each miRNA. The miRNAs with a high PD (mir-92a-3p, miR-486-5p, miR-29a-3p), showed most of their area on one side of 0 (Fig. 1a). In addition, mir-92a-3p and miR-486-5p showed a negative direction, while miR-29a-3p showed a positive direction. Figure 1b shows the ROPE region, being a small area in the first three miRNAs. www.nature.com/scientificreports/ Pathway analysis. For the miRNAs with a high directional probability (has-92a-3p, has-486-5p, has-29a-3p), their potential target genes were analysed in order to assess their involvement in the pathology development. Table 4 shows the potential target genes of the selected miRNAs related to AD mechanisms. As can be seen, 112 potential targets were obtained for miRNA hsa-92a-3p, 16 targets for hsa-486-5p, and 88 targets for hsa-29a-3p, with a target score of at least 95. In addition, each of the selected miRNAs regulated several pathways. As can be seen in Fig. 2, the most common pathways were cell signalling and transcription regulation, but also lipid metabolism, protein synthesis and modifications, and structural functions were regulated by the selected miRNAs. First, the main pathways that could be regulated by the miRNA hsa-92a-3p are cell death or autophagy and cell proliferation pathways, and some pathways related to vesicle transport and synaptic transmission. Among the cell death targets, BCL2L11 (BCL2 like 11) is involved in neuronal and lymphocyte apoptosis and G3BP2 (G3BP stress granule assembly factor 2) is involved in stress response. In the cell proliferation pathway, the gene C21orf91   www.nature.com/scientificreports/  www.nature.com/scientificreports/ transcription; and NEFH (neurofilament heavy), which contributes to the maintenance of neuronal structure. In addition, PPCS (phosphopantothenoylcysteine synthetase) could be relevant in the regulation and metabolism of CoenzymeA. Secondly, the main pathways that could be regulated by the miRNA hsa-486-5p are cell signalling, lipid and protein pathways, structural functions and transcription.
Thirdly, the main pathway that could be regulated by the miRNA hsa-29a-3p is the cell proliferation pathway, which involves neurone regeneration and migration trough NAV3 (neuron navigator3), NAV1, and NAV2. Also, ZNF346 (zinc finger protein 346) could act to protect neurons and LIF (LIF, interleukin 6 family cytokine) is involved in neuronal differentiation. In cell signalling pathways, the targets DAAM2 (dishevelled associated activator of morphogenesis 2) and ROBO1 (roundabout guidance receptor 1) contribute to nervous system development and neuronal migration, respectively. Furthermore, miRNA hsa-29a-3p plays a role in structure regulation, specifically regulating the synthesis of different collagen chains, and HMCN1 (hemicentin 1) is involved in macular degeneration and C1QTNF6 (C1q and TNF related 6) is involved in identical protein binding activity. Also, this miRNA could regulate REST in the transcription pathway.

Discussion
In this study, miRNA sequencing was carried out to identify potential early AD biomarkers. From these, a validation step was conducted, in which quantifiable miRNAs were identified, while some of them were not detected. In fact, the miRNAs not validated were hsa-miR-142-5p, hsa-miR-1293 and hsa-miR-4259. A previous study in cell line found a relationship between dysregulation of miR-142-5p expression and AD pathogenesis and synaptic dysfunction 29 , and it was detected up-regulated in the blood of AD patients 30 . Also, hsa-miR-4259 was detected Table 4. Potential target genes and related AD pathways. In this link it can be found the full name of each gene (http:// mirdb. org/ mirdb/ index. html). www.nature.com/scientificreports/ in saliva samples, but there is a lack of studies quantifying this biomarker in plasma samples 31 . In addition, has-miR-1293 was previously detected in platelets from hepatocellular carcinoma and lung adenocarcinoma cell line 32 . Nevertheless, there are no studies describing its association with AD. Regarding the methodology, Haining et al., performed a similar study trying to find a miRNA profile in early AD. However, different cohorts for untargeted and targeted analysis were used 17 . Also, Dakterzada aimed to find miRNAs in plasma from AD participants, identifying a BACE-1 related panel of biomarkers different from the miRNAs in the present work 33 . It could be due to the use of a different identification technique based on microarrays analysis 34 . The different methodologies employed could affect the miRNAs selection, so it should be taken into account in comparisons with other studies 33 .
Regarding the miRNAs that showed a trend with the pathology in the present study, they were hsa-miR-92a-3p, has-miR-486-5p and hsa-miR-29a. First, hsa-miR-92a-3p showed a tendency for decreased levels in AD. A previous study showed dysregulation of 3 miRNAs related to synaptic proteins, including hsa-miR-92a-3p in MCI and AD 35 . Another study described the relationship between miR-92a-3p and tau accumulation 36 . One of the most AD-relevant pathways that could be regulated by this miRNA is synaptic transmission 37 . Specifically, Figure 2. Pathways regulated by the three miRNAs that showed relationship with AD. The arrows indicate those miRNAs involved in each pathway. Each color represents a miRNA: green (hsa-miR-92a-3p), red (hsa-miR-486-5p) and blue (hsa-miR-29a-3p). *Created with BioRender.com. www.nature.com/scientificreports/ SYNJ1, a potential target for this miRNA, seemed to be involved in amyloid beta clearance 38,39 , while synapsins could act on amyloid beta generation by modulating BACE1 40 . In addition, CBLN4 could regulate amyloid beta toxicity 41 . Regarding neuronal apoptosis, it could be regulated by this miRNA and the BCL2L2 target. In fact, a previous work showed that amyloid beta could regulate that pathway 42 . Other target genes (NEFH, REST), which are involved in neuronal structure and neuronal gene transcription, were described as potential AD diagnosis biomarkers 43,44 . Second, the present study showed a tendency towards reduced levels of hsa-miR-486-5p in AD. Similarly, Nagaraj et al. described a panel of 6 plasma miRNAs, including hsa-miR-486-5p, that differentiated between controls and MCI-AD 45 . This miRNA could regulate some genes involved in cell signalling, lipid and protein pathways, transcription and structural function.

Scientific
Third, a trend towards higher levels for hsa-miR-29a-3p in AD plasma was found. Similarly, Shioya et al. described differential levels of this miRNA at brain levels, suggesting its implication in neurodegeneration trough NAV3 (Neurone Navigator 3) regulation 46 . In addition, another miRNA from that family (hsa-miR-29c) has been related to AD pathology due to its involvement in the amyloid beta accumulation through the regulation of BACE1 47,48 . Moreover, Müller et al. suggested that miR-29a could be a candidate biomarker for AD in CSF samples without cells 49 . In this regard, different types of collagenous chains and C1QTNF6 are targets of miRNA hsa-29a-3p. Previous studies described collagenous chains as a component from amyloid plaques 50 . The collagenous regulation may contribute to the assembly of amyloid fibres, enhancing the development of amyloid pathology. In addition, C1q complement protein co-localizes with the amyloid beta in brain 51,52 . Therefore, C1QTNF6, which is thought to play a role in identical protein binding, could help in the accumulation of C1q protein, triggering amyloid plaque formation (PubMed Gene). In addition, ROBO1 and DAAM2, which are involved in neuronal migration and nervous system development, are targets for this miRNA. In fact, ROBO1 could show a relationship with axon guidance dependent on presenilin, which helps in the proteolysis of amyloid-β precursor protein and triggers to AD pathology development 53,54 . Furthermore, DAAM2 was described by Ding et al. as a mediator in regenerative oligodendrocyte differentiation; while Sellers et al. demonstrated that Aβ synaptotoxicity is mediated by this protein 55,56 .
The main limitations in this study are the small sample size, since it is quite difficult to have a large number of biologically classified early AD patients (MCI, preclinical). Moreover, from the selected miRNAs, some of them were not validated as they were not correctly quantified, probably due to the fact that they were detected in few samples. In addition, the study design is cross-sectional. In order to obtain more accurate data from the different disease stages, it should be longitudinal. However, participants in this present study are perfectly characterized according to CSF biomarkers and their cognitive status, providing a reliable approach to the disease progression.

Conclusions
RNA sequencing analysis in plasma samples from participants with early AD and healthy controls allowed to identify some differentially expressed miRNAs. From them, 3 selected miRNAs (miRNA-92a-3p, miRNA-486-5p, miRNA-29a-3p) were slightly dysregulated in AD, being potential biomarkers of the pathology. In fact, they could be involved in the regulations of important pathways of the pathology, such as synaptic transmission, cell signalling, structure maintenance or cell metabolism, so they could be relevant therapeutic targets. However, further research with a larger sample is needed to verify these results, as well as to develop the potential mechanisms of action of these miRNAs.

Data availability
The datasets generated during the current study are available in the ArrayExpress repository, accession number E-MTAB-11103.